HomeFAQsCan I use a 15-nt overlap that is entirely composed of His-tag repeats (i.e., CACCACCACCACCAC)?

FAQ: Can I use a 15-nt overlap that is entirely composed of His-tag repeats (i.e., CACCACCACCACCAC)?

No, you must flank the His-tag sequence on both sides with at least 2 nucleotides that are not part of the His-tag repeating sequence. Alternatively, intersperse CAC and CAT his codons to interrupt this repetitive sequence. You should avoid repeating sequences at the end of an overlap.

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NEBuilder Assembly Tool can be used to design primers for your NEBuilder HiFi or Gibson Assembly reactions based on entered fragment sequences and the polymerase being used for amplification. This video will highlight some useful updates and the main differences between the two versions.